It is widely desirable to provide rapid, effective detection and identification of various and numerous microorganisms in test samples, say for example, from, but not limited to, water, food and body fluids, such as for example, to detect and identify a total coliform bacteria and/or also E. coli bacteria in a particular test sample.
One enzymatic test identification method is known as the MUG test which was designed for the detection of E. coli. This test is well known and is set forth in U.S. Pat. No. 5,223,402, hereby incorporated by reference. The test method is for detecting total coliform bacteria and E. coli and other microbes employing one or more chemiluminescent compounds in an enzymatic test technique. The test is the detection of a qualitative presence or absence of total coliform bacteria or E. coli or other microbes in a sample by combining the sample with a 1-2-dioxetane compound which decomposes to a light-emitting portion with the reaction of at least one hydrolytic enzyme present in the microorganism in the sample, thereby triggering light emission, so that the light emission can then be detected. The test indicates the presence of the hydrolase activity of the particular microbes in the sample on exposing the light-emitting sample to a light-sensitive detector over a period of time.
An improved MUG test method is directed to the simultaneous detection of total coliform bacteria and E. coli in a test sample, for example a water sample, in a test method known as Fluorocult.RTM. LMX Broth (a culture medium trademark of BDH Inc. of Brampton, Ontario, Canada). This improved MUG test method is described for example in the paper "Simultaneous Detection of Total Coliforms and E. coli--Fluorocult.RTM. LMX Broth" by Dr. Rolf Ossmer et al presented at the 15th International Symposium/FOOD MICRO 1993, The International Committee on Food Microbiology and Hygiene, Aug. 31-Sep. 3, 1993 in Bingen/Rhine, Germany, hereby incorporated by reference.
The improved MUG test provides for a selected enrichment broth which permits the simultaneous detection in the qualitative manner of total coliforms and E. coli in bacterial testing of water, food and other materials. The broth has been formulated to provide a high nutritional quality and phosphate buffers to guarantee a high growth rate for the coliforms present and employs a lauryl sulfate to inhibit to a large extent the growth of gram-positive bacteria. The simultaneous detection of total coliforms and E. coli are made possible by the addition of chromogenic substrates in the broth which permit the easier identification of coliforms due to a color change from a yellow color to a blue-green color about the coliforms. The use of the MUG compound provides more specific identification of E. coli within the test sample. The broth employs a halo-indolyl-.beta.-D-galactopyranoside (X-GAL) which is a 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside which is cleaved by coliforms producing a blue-green color in the broth after incubation. The visual observation of this blue-green coloration would indicate the presence of total coliform bacteria in the test sample, while the absence thereof indicates the absence of total coliform bacteria to a lower limit.
The broth also employs an amplification agent, such as a thiogalactopyranoside, such as a 1-isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG) for an amplification factor in the enzyme synthesis and increases the activity of the .beta.-D-galactopyranoside base. The MUG agent which is a fluorogenic substrate is an alkyl-umbelliferyl-.beta.-D-glucuronide, in particular, a 4-methylumbelliferyl-.beta.-D-glucuronide (MUG), which is cleaved by the enzyme .beta.-D-glucuronidase, which is highly specific for E. coli.
The detection of E. coli is determined by measuring fluorometrically in the long-wave UV range, which fluorescence indicates the presence of E. coli and the absence of fluorescence indicates the absence of E. coli in the test sample. The test broth employs a tryptophan concentration to improve the indolyl reaction for additional confirmation of E. coli and increases the sensitivity of detection in combination with the X-GAL and the MUG reaction. In the prior art, a typical broth composition would then include tryptose, sodium chloride, sorbitol for sugar fermentation, tryptophan, di-potassium hydrogen phosphate and potassium dihydrogen phosphate, lauryl sulfate sodium salt and X-GAL, MUG and IPTG. Thus, the Fluorocult.RTM. test permits the determination of the presence or absence of total coliform bacteria, Esherchia, Enterobacter, Klebsiella and Citrobacter as well as E. coli bacteria.
The Fluorocult.RTM. LMX Broth in use provides a single strength preparation which is a dehydrated culture medium which is then added to water and subsequently poured into a test tube or test container and autoclaved for 15 minutes at 121.degree. C. Ideally, the LMX Broth should have a pH of 6.8.+-.0.1 at 25.degree. C., and the prepared broth is generally clear and either colorless or slightly yellow. The test sample is added to the prepared broth and incubation is carried out for 24 hours, and in some cases 48 hours, at 35.degree. C. to 37.degree. C. The presence of coliforms is determined by the broth turning blue-green due to the X-GAL reaction while E. coli is detected by measuring the fluorescence which is represented by a light blue fluorescence in the broth. The presence of E. coli may also be confirmed by covering the culture with KOVACS indole reagent, and the presence of E. coli detected by a cherry red color appearing in the reagent, later after one or two minutes, to confirm the presence of E. coli if desired.
None of the cited references are suitable to determine bacteria quantitatively in a large volume of a sample, for example, about 10 ml to 500 ml as in water samples. In order to enumerate bacteria in large sample volumes, e.g. over 10 ml, the sample needs to be filtered through a 0.22-0.45 .mu.m filter or to use multiwell plates.
The filtration technique is the standard technique being used by regulatory agencies and microbiological analytical labs to numerate coliforms in 100 ml samples. Sample filtration with a 0.45 .mu.m filter is done first to recover bacteria from 10 ml to 300 ml water or extraction solution (from particulate food or soil sample) and then cultivate the filter (with the bacteria) on an agar plate with growth media, such as the LMX Fluorocult.RTM. or conventional media for coliforms, such as the M-endo agar. This technique needs a sterile filtration assembly, a microbiological hood to perform the filtration and preparation of agar plates. It is laborious and needs a laboratory support, e.g. autoclave, hood and vacuum system for filtration. Common problems encountered with this filtration technique are from small particulates that block the filter, such as silt, dust, rust or other suspended particulates.
Another method is using specific antibodies immobilized to plastic beads or magnetic beads for specific recovery of target bacteria, followed by cultivation of the beads in growth media or selective growth media. This antibody method is expensive and again requires work under sterile conditions and needs highly trained laboratory personnel.
A metabolic identification method is also common and used by microbiological labs and commercial companies for identification of thousands of microorganisms. This method requires enrichments and purification of cultures (a single colony), and it is a laborious, multistep procedure which can take 48 to 96 hours for identification of individual bacteria. It has been reported by Biolog, Inc. of Hayward, Calif. that over 1,100 species can be identified by metabolic tests using specific enzymes and substrates utilization. However, the method is expensive and requires highly trained and skilled laboratory personnel.
Another method known as the Colilert.TM. (a trademark of Idexx Laboratories, Inc. of Westbrook, Me.) method uses substrate technology of an Idexx Quanti-Tray for coliform enumeration in 100 ml water samples. However, this method which is routinely used by the biotechnology industry for isolation of bacterial or transformed cells is costly, and in the specific case of the Colilert.TM. method requires a heat sealing system. Numeration at the 25-100 cfu becomes a problem as multiple bacteria can grow in a single cell, and a complex mathematical model is used to correlate the visual results with the actual count (Most Probable Number statistical model--MPN). The Quanti-Tray sealer required for this operation can also become a source of cross contamination. This method is described and claimed in WO95/23026, published Aug. 31, 1995.
It is desirable to provide a new and improved bacteria, test particularly for coliform/E. coli that is simple to be performed by the layman, without laboratory equipment and still gives qualitative as well as quantitative results in a short period of time. For example, a test can be performed at 35.degree. C. to 45.degree. C. for 16-24 hours (incubator required) or at a lower temperature of 20.degree. C. to 30.degree. C. for 48-72 hours.
It is desirable to provide an improved MUG test wherein the MUG test either for total coliform bacteria alone or E. coli, or a combination thereof, may be rapidly and effectively, not only qualitatively, but quantitatively, determined.